The present invention relates to a method and apparatus for maintenance and expansion of hemopoietic stem cells. More particularly, the present invention relates to a three dimensional stromal cell plug flow bioreactor for the maintenance and/or expansion of hemopoietic stem cells and/or for the production of a conditioned medium for the maintenance and/or expansion of hemopoietic stem cells.
The hemopoietic system in mammals is composed of a heterogenous population of cells that range in function from mature cells with limited proliferative potential to pluripotent stem cells with extensive proliferative, differentiative and self renewal capacities (1-3). Hemopoietic stem cells (HSC) are exclusively required for hemopoietic reconstitution following transplantation and serve as a primary target for gene therapy. In spite of the key role of stem cells in maintaining the hemopoietic system, their extremely low frequency in hemopoietic tissue, as well as the limited ability to maintain or expand undifferentiated stem cells under ex-vivo conditions for prolonged periods of time, not only remains a major drawback to essential clinical applications of these cells, but also reflects the current unavailability of, and the need for, novel stem cell regulators.
It is widely accepted that stem cells are intimately associated in vivo with discrete niches within the marrow (4-6), which provide molecular signals that collectively mediate their differentiation and self renewal, via cell-cell contacts or short-range interactions (7). These niches are part of the “hemopoietic inductive microenvironment” (HIM), composed of marrow stromal cells' e.g., macrophages, fibroblasts, adipocytes and endothelial cells (8). Marrow stromal cells maintain the functional integrity of the HIM by providing extracellular matrix (ECM) proteins and basement membrane components that facilitate cell-cell contact (9-11). They also provide various soluble or resident cytokines needed for controlled hemopoietic cell differentiation and proliferation (12-14).
In view of the above, it is not surprising that efforts to develop culture systems for the prolonged maintenance of human HSC were mainly focused on the use of pre-established primary marrow stromal cell monolayers. These included long-term cultures of unirradiated (Dexter cultures, 15) or irradiated (16-19) primary human marrow stroma, as well as human or murine stromal cell lines (16, 19-24), with or without exogenously added cytokines. Output assay for HSC initially relied on the capacity of such cells to produce myeloid progeny (long-term culture initiating cells; LTC-IC) or to generate colonies with cobblestone morphology (cobblestone area forming cells; CAFC) after prolonged culture (5-7 weeks) on such stromal cells (16,17). In spite of the widespread use of LTC-IC and CAFC assays, it is becoming increasingly obvious, however, that they detect highly primitive progenitors, rather than true repopulating hemopoietic stem cells (25, 26).
A recently developed human stem cell assay detects a SCID repopulating cell (SRC), which homes to the bone marrow of non-obese diabetic (NOD)/SCID mice (27), where it gives rise to human myeloid, lymphoid, erythroid and CD34+ progenitor populations (28-30). The SRC is exclusively found in hemopoietic cell fractions expressing the CD34+38− surface antigen (31) and its frequency in CB (1/3×105 cells) is enriched as compared to BM (1/9×105 cells) or mobilized PB (1/6×106 cells) (32). Very recent studies showed that the SRC resides-within a subpopulation of CD34+/38−/CXCR4+ cells (33). CXCR4, a surface receptor for the chermokine stromal cell-derived factor 1 (SDF-1, 34), is apparently essential for homing and engraftment of human hemopoietic stem cells in the NOD/SCID marrow (33).
Studies aimed at inducing prolonged maintenance/expansion of human HSC on stromal cell cultures were mainly based on CAFC, LTC-IC or the CD34+38− phenotype as end-point assays (16, 19-24). Rare reports of SRC maintenance/expansion in stromal cell cultures fail to indicate significant long-term support. For example, allogeneic human marrow stroma was found to induce short-term (7-day) SRC maintenance, followed by a rapid, marked decline (6-fold) in activity (26). The inability to support the long-term maintenance/expansion of transplantable human stem cells on stromal cell layers, may be attributed to several factors related to in vitro cultures of these cells. Among these, one may include the use of stromal cell monolayers, which do not reflect the in vivo growth conditions within the natural, three-dimensional structure of the bone marrow. Such conditions may diminish the capacity of stromal cells to provide the optimal, appropriate supportive microenvironment, as well as the capacity of stem cells to localize in specific niches and to physically interact with stromal cells and their products. Indeed, evidence for the importance of a three dimensional (3D) structure for the biological activity of hemopoietic progenitor cells, is provided by the superior growth of a human hemopoietic cell line on stromal cells seeded in a 3D collagen matrix, as compared to their proliferation on monolayers of such cells (35). More importantly, a 3D tantalum-coated porous biomaterial, was recently shown to enhance the short-term maintenance of macaque LTCIC or CD34+38− cells, as compared, to cells cultured alone or on marrow stromal cell monolayers (36). The effect of stromal cell-coated 3D carriers, was, however, not investigated.
Recent studies have shown the murine AFT024 cell line to be superior than human stroma, in supporting 2-3 week survival and maintenance (albeit not expansion) of human CB SRC (37). This line has been found to express several novel HIM genes encoding membrane-bound proteins (21, 38, 39), which may have an essential role in stem cell physiology. The possible expression of these and other genes by stromal cells under conditions which more closely mimic their 3D marrow microenvironment, and thus enable their optimal, physiological functional activity, has yet to be determined.
Extensive studies have shown that stroma non-contact cultures (19, 21, 22, 40, 41) or stroma conditioned media (SCM) (21, 42-44), alone or with cytokines, can support the ex-vivo maintenance or expansion of primitive hemopoietic progenitors. SCM has also been shown to improve the recovery and transduction efficiency of such cells (45, 46). While these findings again stress the importance of soluble stromal cell factors, the use of LTC-IC, CAFC or CD34+38− end-points in such assays cannot reflect the effect of SCM on maintenance/expansion of transplantable HSC. Furthermore, it is not known whether such SCM, obtained from monolayer cultures of stromal cells, indeed contains all stromal cell-associated gene products involved in human HSC physiology.
Recent attention aimed at ex-vivo expansion of transplantable hemopoietic stem cells has focused on the establishment of cytokine-supplemented suspension cultures (47-53). These studies have helped identify the major relevant cytokines' for this process, e.g., early-acting ones such as stem cell factor (SCF), FLT3 ligand and thrombopoietin (TPO). Nevertheless, variable results have been obtained, indicating short-term loss (48, 49), maintenance (50-52) but also some rare examples of SRC expansion, following during 2-4 weeks of culture (47, 53). The interactive capacity of these cytokines and stromal cells, under 3D growth conditions, to support the maintenance/expansion of SRC, has not yet been defined.
There is thus a widely recognized need for, and, it would be highly advantageous to have, a method and apparatus for ex-vivo expansion and/or maintenance of transplantable hemopoietic stem cells devoid of the above limitations, with superior results as is compared to the teachings of the prior art.